Immunological assay methods

ABSTRACT

Immunoassays of psychoactive drugs including psychotomimetic drugs, narcotic drugs, and tetrahydrocannabinols and treatment methods based on the antigenic properties of protein conjugates of these drugs. These methods are based upon treating the psychoactive substances as haptens and utilizing their protein conjugates to produce antibodies to the psychoactive materials themselves. The immunoassay methods include both agglutination and agglutination-inhibition reactions. The treatment methods include treatment or both exogenous, administered drugs (such as cannabinols, LSD, heroin and morphine) and endogenous substances (such as N,N-Dimethyltryptamine and 5-Methoxy-N,N-Dimethyltryptamine by active immunization and also passive immunization.

This is division of application Ser. No. 699,159, filed on May 13, 1991,which is a division of application Ser. No. 345,964, filed May 1, 1989,now U.S. Pat. No. 5,037,645, which is a division of application Ser. No.925,821, filed Oct. 30, 1986, now U.S. Pat. No. 4,834,973, which is adivision of application Ser. No. 319,238, filed Nov. 9, 1981, now U.S.Pat. No. 4,620,977, which is a continuation of application Ser. No.761,290, filed Jan. 21, 1977, now U.S. Pat. No. 4,375,414, which is acontinuation of application Ser. No. 255,154, filed May 19, 1972,abandoned.

BACKGROUND OF THE INVENTION

This invention relates to improved immunoassays of psychotomimeticdrugs, narcotic drugs, tetrahydrocannabinols and other psychoactivedrugs.

At the present time, there are certain methods used for thedetermination of psychotomimetic and narcotic drugs in biologicalmaterials.

The techniques that are used in the present time for the determinationof drugs in biological materials, are described in detail in theHandbook of Analytical Toxicology (Irving Sunshine, Editor; The ChemicalRubber Company, Publisher; Cleveland, Ohio, 1969). They include indifferent combination for the different drugs: paper, thin layer andgas-liquid chromatographic methods, crystal tests, fluorescence,infrared, ultraviolet, thermal microscopy and animal pharmacologystudies.

In general, the tests are time consuming, expensive, require expensiveequipment, and require well trained personnel. Some of the tests are notsensitive, others lack high specificity. Special difficulty isencountered in the determination of one drug in the presence of otherdrugs in the same biological material specimen. Thus heroin is difficultto determine in the urine in the presence of nicotine, as disclosed byD. J. Berry et al in "The Detection of Drugs of Dependence in Urine"(Bulletin on Narcotics 22, No. 3, July-September 1970; United NationsPublication). Tetrahydrocannabinols are difficult to determine in thepresence of barbiturates, and complicated methods are needed for theirdetermination in the presence of barbiturates, as described by Harold V.Street in "Identification of Drugs by a Combination of Gas-Liquid, Paperand Thin-Layer Chromatography" (Journal of Chromatography 48,291-4,1970).

The methods of the present invention have the advantages of simplicity,speed, specificity and low cost. They also have the advantage of beingable to be applied "on the spot" (e.g. emergency room of a small fieldhospital). Van Vunakis et al ("Production and Specificity of AntibodiesDirected towards 3,4,5-Trimethoxy-phenyl-ethylamine, and2,5-Dimethoxy-4-methylamphetamine," Bioch. Pharmacol. 18, 393-404, 1969)were able to obtain high specificity and sensitivity in theirdetermination, by microcomplement fixation inhibition of3,4,5-Trimethoxy-phenylethylamine and congeners, as well as2,5-Dimethoxy-4-Methylamphetamine and congeners.Micro-complement-fixation-inhibition is however a complicated method.Reagents have to be prepared freshly for each experiment, and theyrequire specially trained personnel. In the methods hereinafter to bedescribed, no such limitations are present.

The length of time required by presently known procedures fordetermining psychoactive drugs also severely limits their usefulness inclinical applications.

The invention also includes immunological methods for the treatment ofdrug intoxication; the treatment and prevention of drug addiction, drugdependence and drug abuse; and the treatment of schizophrenia. The needfor treatment methods for intoxication by psychoactive drugs, methodsfor freeing persons dependent on such drugs from their dependence, andmethods of treating schizophrenia has long been felt. The presentmethods provide attractive and useful approaches to all of these needs.

SUMMARY OF THE INVENTION

In accordance with this invention, generally stated, diagnostic andtreatment methods are provided by the use of the haptenic properties ofpsychoactive material. The term "psychoactive" includes psychotomimeticcompounds containing an indol ring such as N,N-Dimethyltryptamine andits congeners and LSD 25 and its congeners; amphetamines and theircongeners; narcotics such as phenanthrene alkaloids (such as morphine,heroin, codeine, hydromorphone, and levorphanol) and nonphenanthrenealkaloids (such as meperidine, methadone, and phenazocine); andtetrahydrocannabinols and other cannabinoids.

A hapten may be defined as any small molecule which by itself does notproduce antibodies but which, when conjugated to a carrier protein orother macro-molecular carrier, induces in the recipient animal or humanthe production of antibodies which are specific to the small molecule.

The present invention is based in part upon the application of knownimmunoassay techniques for haptens to certain psychoactive compoundswhich have not heretofore been recognized as haptens (such asN,N-dimethyltryptamine and congeners, and tetrahydrocannabinols andtheir congeners); in part upon the discovery of methods of adaptingtechniques which were heretofore used only for the determination ofantibodies or complete antigens to techniques for determining haptens;in part upon the discovery of immunological treatment methods for suchseemingly disparate medical problems as drug intoxication, drugdependence and schizophrenia; and in part upon the development ofentirely new methods for the treatment of drug intoxication based inpart upon the haptenic characteristics of the intoxicating drugs.

The discovery that 5-methoxy-N,N-dimethyltryptamine and congeners arehaptens and the recognition that tetrahydrocannabinols(such asdelta-9-tetrahydrocannabinol) are haptens, permits their determinationby known immunoassay methods for haptens, such as radioimmunoassay(Spector et al, Science 168, page 1347, 1970; Niswender et al, inImmunological Methods in Steroid Determination, edited by Peron andCaldwell, 1970, pages 149-173) and micro-complement fixation inhibition(Levine, in Handbook of Experimental Immunology, edited by D. M. Weir,1967, pages 707-719, especially page 712). These psychoactive haptensalso may be utilized in the determination of their antibodies by methodssuch as those described in Handbook of Experimental Immunology (ed.Weir), pages 423-968, for example hemagglutination (W. J. Herbert"Passive Hemagglutination" in Handbook of Experimental Immunology, pages720-744).

The invention also encompasses the determination of haptens, andparticularly psychoactive haptens, by simple and accurate agglutinationand agglutination-inhibition assays.

The agglutination inhibition assay includes the steps of mixing a samplecontaining an unknown quantity of psychoactive hapten with apredetermined quantity of an antibody to the hapten, and then combiningthis mixture with a predetermined quantity of the hapten bound to anagglutinable particulate carrier. Presence of a sufficient amount of thepsychoactive hapten in the sample will inhibit hemagglutination. Theusual tray or other equipment may be utilized to obtain a quantitivemeasure of the psychoactive hapten (Handbook of Experimental Immunology,pp 782-785).

The agglutination methods involve the binding of an antibody to anagglutinable particulate carrier. This binding may require the use of achemical binding technique such as the bis-diazotized-benzidine (BDB)technique. These techniques are set out in Handbook of ExperimentalImmunology, pages 737-740, Cua-Lim et al, J. Allergy 34, 142 (1963);Ingraham, Proc. Soc. Exp. Biol., N.Y. volume 99, 452 (1958).

These agglutinable carrier-bound antibodies may be used in a number ofagglutination procedures. In one, a sample is mixed with theagglutinable carrier-bound antibody in suspension, the agglutinablecarrier-antibody is then mixed with a free antibody to the hapten. Ifthe sample contains above a minimal amount of the free hapten,agglutination will result. In another method antibodies to two differentsites on the hapten are prepared and bound to an agglutinableparticulate carrier. Addition of a sample which contains the free haptenproduces agglutination. Other procedures utilizing two differentantibodies to two different sites on the hapten may be provided in whichonly one, or neither, of the antibodies is bound to an agglutinableparticulate carrier.

In all of the agglutination and agglutination inhibition procedures,erythrocytes (red blood cells) are the presently preferred carrier. Theerythrocyte may or may not be treated, for example by formalin treatment(Ingraham, supra). However, other agglutinable material such as latex orother particles may be useful in some or all of the methods.

The invention also encompasses the passive immunization for drug(hapten) intoxication. It is particularly directed to in vitroimmunotherapy methods through immuno-dialysis or immuno-adsorbtiontechniques. It also may be applied to auto-intoxication by substancesetiological to diseases such as schizophrenia. For example, recentresearch indicates that N,N-dimethylated-indoleamine are psychotomimeticagents which are etiological to schizophrenia. See for example Tanimukaiet al in Recent Advances in Biological Psychiatry, volume 10, pages 6-15(1968); Narasimhachari et al in Biological Psychiatry volume 3, pages21-23 (1971). Therefore, one treatment method includes the passiveimmunization of schizophrenic patients with antibodies toN,N-dimethylated-indoleamines. Antibody fragments may also be used. Thetreatment of species which are etiological to schizophrenia and also arecomplete antigens may include the step of splitting the antibodies (forexample by the method set out in Boyd, Fundamentals of Immunology (4thedition, 1966), especially at pages 70-71) in order to preventprecipitation of the species. The active immunization procedure may beused both in schizophrenia, by administration of an antigen whichproduces antibodies to a species which is etiological to schizophrenia(for example a protein conjugate of N,N-dimethyltryptamine), or in thetreatment of drug dependent persons. The treatment includesadministration of an antigen (or antigens) which produces antibodies tothe drug (or drugs) on which the individual is dependent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a hemagglutination inhibitionprocedure utilized in the methods of the present invention.

FIG. 2 is a diagrammatic representation of a test system of Examples 3and 4 of the present invention.

FIG. 2A is a diagrammatic representation of a test system of Examples5-7 of the present invention.

FIG. 3 is a diagrammatic representation of a hemagglutination reactionof the test system of Example 5 of the present invention.

FIG. 4 is a diagrammatic representation of an agglutination reaction ofa test system of Example 8 of the present invention.

FIG. 5 is a diagrammatic representation of a precipitation reaction of atest system of Example 8 of the present invention.

FIG. 6 is a diagrammatic representation of an immunodialysis system ofExample 12 of the present invention.

FIG. 7 is a diagrammatic representation of an immunoadsorption system ofExample 13 of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are illustrative of the methods and materials ofthis invention.

The following methods are utilized to produce the materials needed tocarry out the invention:

a. Conjugation

The psychoactive haptens which contain an immino (NH) group (such as theN,N-dimethylated indolamines and LSD) are conjugated to Human SerumAlbumin (HSA) using the Mannich formaldehyde reaction, as described byRanadive and Sehon, Canad. J. Biochem, 45, p 1701 (1967). Theconjugation in this method is to the immino (NH) group. Otherhapten-macromolecular conjugates are formed by standard methods, forexample those set out in Spector, in Advances in BiochemicalPsychopharmacology, volume 1 (ed. Costa and Greengard) p 181 (1969);Pinckard et al in Handbook of Experimental Immunology, p 493; andGoodfriend et al in Immunologic Methods in Steroid Determination, p 15;and Ingraham, supra.

Determination of conjugation is done following the method described inStrahilevitz et al, Biological Psychiatry 3, 227 (1971), by aspectrofluorometric method,

b. Production of Antibodies to. Haptens

Immunizations of rabbits with conjugates in complete Freund's Adjuvantare carried out by a similar procedure to the one described byStrahilevitz et al, supra. The preparation of antisera and globulinfractions are also described in this reference. The antibodies may bepurified by known methods such as those set out in Immunologic Methodsin Steroid Determination.

c. Preparation of Erythrocytes (Agglutinable Particulate Carrier)

Preparation of formalinized, type O human erythrocytes is done accordingto the methods described by Ingraham, supra. Some cells prepared by thismethod can be used even 15 months after preparation.

d. Binding of Haptens to Erythrocytes

Conjugation of haptens to the formalinized erythrocytes is done by theBis-Diazotized-Benzidine technique (BDB technique).

Determination of presence of antibodies in the rabbit serum anddetermination of their specificity and cross reactivity in the serum ofthe drug-HSA-conjugate immunized rabbits is done by hemagglutination ofthe drug bound erythrocytes by their specific antisera and by specificinhibition of the hemagglutination in the presence of the free drug(hapten inhibition in the presence of the free drug) (see Fultrope etal, Brit. Med. J., Apr. 20, 1963, pp 1049-54). The hemagglutinationprocedure is shown diagrammatically in FIG. 1. For example, inhibitionof hemagglutination by NNDMT but not by other drugs will indicatepresence of antibody specific to NNDMT. Determination of presence ofspecific antibodies is also done by double diffusion and doublediffusion inhibition studies (i.e. inhibition of the precipitation inthe presence of the free drug as described by Ranadive and Sehon, supra,for Serotonin).

EXAMPLE 1: Radioimmunoassay of Tetrahydrocannabinols

A tetrahydrocannabinol is conjugated to a protein by one of the standardmethods utilized to bind steroids to proteins (Immunologic Methods inSteroid Determination). The conjugate is prepared in such a way as toproduce a ratio of tetrahydrocannabinol to carrier molecule of from 4:1to 30:1. The conjugate is emulsified in a phosphate buffer saline (pH7.4). Rabbits, sheep or other suitable animals are immunized with fromabout 1/2-2 mg of the conjugate per kilogram body weight (inconcentration of about 5 mg/ml PBS).in equal volume mixture withcomplete Freund's adjuvant. The mixture is again emulsified beforeinjection into multiple sites, e.g. intradermally, into the foot pad,subcutaneously, and intraperitoneally. Injections are made once everytwo weeks and blood taken from the animal, allowed to clot, and testedfor antibodies, as set out in Ranadive and Sehon, supra.

Radioactively labeled (³ H) tetrahydrocannabinol (obtained from NationalInstitute of Mental Health) in a predetermined amount, is added to theantibody to the tetrahydrocannabinol. Ammonium sulfate (or anotherprecipitating reagent) is then added in order to precipitate thetetrahydrocannabinol-bound antibody. The precipitate is sedimented bycentrifugation and washed. The precipitate is then dissolved with asuitable solubilizing reagent (such as Tween 80 or NCS solubilizer[Amersham/Searle]). A standard curve is prepared by adding to the testsolution varying amounts of unlabeled tetrahydrocannabinol to thepredetermined amount of labeled tetrahydrocannabinol. The quantity oftetrahydrocannabinol in a sample is then determined by addition of thesample (e.g. serum) together with the predetermined quantity of labeledtetrahydrocannabinol and antibody. Comparison of the radioactivity countof the precipitate with the standard curve yields the amount oftetrahydrocannabinol in the sample.

EXAMPLE 2: Radioimmunoassay of 5-methoxy-N,N-dimethyltryptamine

Antibodies to 5-methoxy-N,N-dimethyltryptamine (5-MeO-N-DMT) areprepared according to the method of Strahilevitz et al, supra.5-MeO-N-DMT is conjugated to a radioactive (¹³¹ I) protein.Radioimmunoassays are carried out using the radioactive hapten-proteinconjugate, in the same manner as in EXAMPLE 1.

In EXAMPLES 1 and 2 radioimmunoassay can be modified by using the"double antibody procedure" as described by Niswender et al inImmunological Methods in Steroids Determination, p149. In EXAMPLE 1,non-labeled tetrahydrocannabinol may be conjugated to ¹³¹ I-rabbit serumalbumin, or to another radioactive labeled protein carrier for thepurpose of being used in the radioimmunoassay procedure instead of theradioactive ³ H tetrahydrocannabinol.

EXAMPLE 3: Detection of a Specific Psychoactive Drug by a QualitativeTest

By titration, a suitable amount of antiserum specific to a drug and redcells to which this drug is conjugated, is determined. As shown in FIG.2, tubes 20 made of plastic with a thin plastic divider 21 are used forthis test system. If the drug in question is present in the sample thatis being examined, as a free hapten, it will combine to the antibodiesagainst it after the sample is introduced into the upper compartment ofthe tube and incubated for 15 minutes at 37° c. When, after incubationas described above, the thin plastic barrier 21 in the middle of thetube 20 is broken by the needle with which the sample was put into theupper compartment 22, and the mixture of serum sample tested and thedrug specific antiserum is allowed to mix with the drug-conjugatederythrocytes in the lower compartment of the tube, the presence of atleast a particular quantity of the drug (or a particular range ofquantities) will inhibit or prevent agglutination of the drug-conjugatederythrocytes. Thus agglutination inhibition will be an indication of thepresence of free drug in the sample tested. (If the drug is carried inthe serum, on a macromolecule like protein, or another macromolecularcarrier, then it will be a complete antigen and precipitation may occurwhile the drug is incubating in the upper compartment 22 of the tubewith the specific antiserum. However, hemagglutination inhibition shouldin addition be demonstrated in the lower compartment 23 of the tube.)

EXAMPLE 4: Quantitive Determination of Drugs

This is done with a tray similar to the one that is used routinely formicrohemagglutination studies (see Tyrell, supra).

Serial dilutions of the tested serum is added to the upper compartmentby a small syringe. The difference between the tray used for this testand the one that is being used routinely, is that the tray with thecylinders will be higher, and that each cylinder has an upper and alower compartment divided by a thin plastic divider (as shownschematically in FIG. 2). The procedure is otherwise identical with theone previously described in EXAMPLE 3. The drug "titer" is the highestdilution in which hemagglutination will be inhibited.

Quantitive determination may also be done by the use of routinely usedtrays and loops used for microhemagglutination and microhemagglutinationinhibition.

This method can be used for the determination of a wide variety ofpsychoactive haptens, including the N,N-dimethylated indolamines, LSD,narcotics (like heroin or morphine) and tetrahydrocannabinols, and mayin principle be applied to determining any haptenic chemical or normalor pathological haptenic body metabolite.

In the above described quantitive methods, with each unknown serumsample that will be tested for the presence of drugs, a known controlnegative sample, and a known positive sample, that will include a knownamount of drug, will be done simultaneously.

EXAMPLE 5: Qualitative Determination of Drug-Hapten by Reversed BDBTechnique

Presence of Drug-Hapten in this system will be detected byhemagglutination.

As shown in FIG. 2A, tubes 20 are the same as for EXAMPLE 3. Antibody tothe drug-hapten is prepared in an identical way as described in EXAMPLES3 and 4. The anti drug-hapten antibodies are conjugated to theformalinized erythrocytes as described by Cua-Lim et al, supra, in theReversed BDB technique. They are then placed in the upper compartment 22of the tube. Free antibodies to the drug-hapten are included in thelower compartment 23 of the tube 20. The material tested for freedrug-hapten is then injected into the upper compartment 22 andincubated. If free drug-hapten is present it will be bound to theanti-drug-hapten-antibodies which are conjugated to the erythrocytes.After the incubation, the tube is centrifuged and the red cells in theupper compartment are washed with Phosphate Buffer Saline PH 7.4 (PBS).The thin plastic divider 21 is then pierced by the needle and thematerial drops into the lower compartment 23. If drug-hapten is presentattached to the anti-drug-hapten-antibodies that are conjugated to theerythrocytes, then hemagglutination appears indicating presence of freedrug in the sample.

This is shown schematically in FIG. 3.

The method has the advantage of indicating the presence of hapten byhemagglutination rather than by hemagglutination-inhibition.

EXAMPLE 6: Quantitative Determination of Drug-Hapten by Reversed BDBTechnique

The same technique described in EXAMPLE 5, is utilized for quantitativedetermination of drug-hapten, utilizing the tray and the other methodsas described in EXAMPLE 4. Here again the presence of free-drug-haptenis indicated by hemagglutination.

Addition of material containing free hapten to the upper compartment 22and then incubated causes the hapten-drug to bind to the formalinizederythrocyte bound antibody, and in the presence of unbound antidrug-hapten antibody, agglutination will take place in the lowercompartment 23.

The foregoing methods (EXAMPLES 3-6) can be adapted for use innon-animal materials such as plant materials and drug samples, as canthe following two EXAMPLES.

EXAMPLE 7: Qualitative Determination for the Presence of One Drug out ofa Group of Drugs

This method utilizes a test system similar to the test system describedin EXAMPLE 5. The system is identical with the exception that in theupper compartment 22, a mixture of several batches of formalinizederythrocytes is present, each batch being conjugated with An antibody toa different drug-hapten.

Similarly the lower compartment 23 contains a mixture of unboundantibodies, identical with those present in the upper compartment 22. Ifthe antibodies, present in a free form in the lower compartment 23 andconjugated to the erythrocytes in the upper compartment 22, are specificagainst drugs: A, B, C, D, E . . . and if any one of these drugs or someof these drugs is present in the tested sample, a positive reaction,indicated by a hemagglutination, will be visible in the lowercompartment 23 thus indicating that one or more of the drugs A, B, C, D,E . . . are present in the sample.

A detailed analysis for specific drugs as described above will determinethe specific drug or drugs which are present in the tested sample.

EXAMPLE 8: Production and Use of Antibodies to Different Sites on aHapten

In the foregoing EXAMPLE, in order to ensure that the hapten will haveat least two combining sites bine with the antibodies, the hapten can beconjugated to the carrier protein (or another macromolecular carrier)through two different sites in the hapten molecule. For example, withestrone, conjugation will be done to rings A and D respectively in theestrone molecule by the methods described by Goodfriend and Sehon inImmunological Methods in Steroid Determination, supra. Conjugation totwo different sites in the hapten with protein, can be done in varioushaptens by a variety of conjugation methods. The two conjugates of thehapten (in the example the protein conjugates of Estrone through ring Aand D respectively) will be used for immunization of animals (such asrabbits). (Immunization with each of the conjugates is preferably donein different rabbits.) It is expected that the immunized rabbit willproduce antibodies directed towards an antigenic site on the estrone orother hapten molecule which is sterically guided by the site on themolecule through which conjugation to the protein was done. Generallythe antibody will be directed toward a site away from (complementary to)the conjugated protein. Besides using different conjugation methods,other methods by which usefully different antibodies to the same haptenmay be produced include the use of antibodies to the conjugate of thehapten with two different carriers, like HSA (human serum albumin) andhemocyanin or other two different carriers. Either the same method ofconjugation or different methods of conjugation can be used forconjugating the hapten to the two different carriers. Each one of thetwo antisera may then be specific against another antigenic site on thehapten molecule. The same result may be obtained or enhanced by activelyimmunizing different animal species (such as sheep and rabbits) with thehapten-protein conjugate or conjugates to produce the first and secondantibodies.

The antibodies to two hapten sites may be used in a number ofprocedures, as follows:

a. Both antibodies are conjugated to red blood cells (RBC) or otheragglutinable-particulate carrier like latex and placed in suspension, asin the preceding example, in a container. As shown in FIG. 4, in thepresence of free hapten in the test material (e.g., free estrone) anantigenic site on the ring A of the estrone will attach itself to theantibody produced against the protein conjugate which was conjugatedwith estrone through ring D. Similarly, an antigenic site on ring D ofestrone will attach itself to the antibody produced against the proteinconjugate which was conjugated with estrone through ring A. Therefore,the presence of at least a certain amount (i.e. a certain range) ofhapten in a sample will produce agglutination.

b. As shown in FIG. 5, with the use of anti A and anti D antibodies inthe test system estrone can be demonstrated by precipitation in fluid orsolid media. Other haptens as well can be determined by precipitation inthe presence of antibodies to two different antigenic sites in thehapten.

c. Other procedures may use combinations of antibodies which are boundto agglutinable particles and antibodies which are not so bound. Also,more than two different antibodies may be used.

All of the foregoing hemagglutination tests can be used in the presenceof developing agents such as polyvinylpyrolidone (P.V.P.) or Dextran inorder to increase their sensitivity. (Handbook of ExperimentalImmunology, p. 995).

EXAMPLE 9: Active Immunization Treatment of Schizophrenia

Active immunization treatment of schizophrenia is carried out with aconjugate of a psychotomimetic compound (or a combination of conjugatesof such compounds) such as N,N-dimethyltryptamine,5-methoxy-N,N-dimethyltryptamine, tryptamine, and other psychotomimetichaptens, the presence of which has been described in the biologicalmaterials of schizophrenic individuals. These psychotomimetic smallmolecules are conjugated with protein macromolecular carrier, or withpolyamine acid molecules as carriers (like: polylysine or polyarginine).

Immunization of the patient is carried out either without the use ofadjuvant or with the use of an adjuvant. This will be tried in humansonly after the safety of the compound, safety of the route ofimmunization, schedule of immunization, and the safety of the adjuvantused if any, has been extensively studied, and determined in studies inexperimental animals, by suitable methodology which is available at thepresent time.

Treatment of schizophrenia by active immunization with complete antigens(immunogens) that may be etiological in the disease (either protein orother complete antigens (immunogens)) will include immunization withenzymes that may be etiological in schizophrenia like: indoleamine Nmethyl transferase, indoleamine O methyl transferase, catechol amine Nmethyl transferase, catechol amine O methyl transferase as well as otherenzymes that may be related to the etiology of schizophrenia.

EXAMPLE 10: Treatment of Schizophrenia by a Passive Immunotherapy Method

Immunization of the patient is carried out by administering antibodiesto species which are etiological to the disease. For example, in acuteschizophrenia, antibodies to 5-methoxy-N,N-dimethyltryptamine may beadministered.

In passive immunization treatment with antibodies against completeantigens present in schizophrenia serum, in order to avoid possibleprecipitation of the complete antigens by the antibodies, the anitbodiesmay be treated in such a way as to make them "univalent", for example bymild reduction with dilute HCl and breaking of the antibody moleculeinto two identical halls each with one combining site (William C. Boyd,Fundamentals of Immunology 4th edition, Interscience Publishers NewYork, London, Sidney, 1966 pp 70,71). Other methods for obtaining"univalent" non-precipitating antibody, that can yet bind the antigenand possibly neutralize it are also summarized in this reference and canbe utilized.

EXAMPLE 11: Treatment of Drug Dependence by Active Immunization

The immunization of drug dependent individuals is in principle similarto the immunotherapy of schizophrenia by active immunization withprotein conjugates of methylated indoleamines. The drug or drugs onwhich the individual is dependent are conjugated to a protein or othermacromolecular carrier, for example by one of the methods discussedhereinabove. After the individual is withdrawn from the drug (forexample by methadone withdrawal, for heroin), he will be immunized byadministration of the conjugate, in a manner similar to that used forthe active immunization treatment of schizophrenia.

Many conditions require the removal of species from the blood of anindividual under circumstances which make active immunization and evenpassive immunization treatments (as well as other treatment methods)impractical. Examples of such conditions are severe intoxication (suchas with phenothiazines) and certain acute schizophrenic conditions (suchas acute catatonic stupor). In addition, methods are needed for removal,from the circulation of a patient having a malignant tumor, of certainantibodies against tumor-specific antigens. These specific antibodiesare known to be "enhancing antibodies" (which stimulate the growth ofthe tumor). Removal of these antibodies may be therapeutic for suchpatients. The removal of antibodies which may be etiological in certainallergic conditions or autoimmune diseases is expected to be therapeuticfor these conditions. These antibodies include the antibodies specificto the septal region of the brain, e.g. "Taraxein", which according tofindings of Heath et al (Am. J. Psychiat., 124, p 1019,(1968); Arch Gen.Psychiat., 16, pp1 (1967)) are etiological in schizophrenia. It also maybe desirable to remove from the circulation of schizophrenic patientsother proteins such as the alpha-2 globulin which was found by Frohmanet al (Ann. N.Y. Acad. Sci., 96 p 438 (1962)) in schizophrenic patientsand which may be etiological in schizophrenia. In all of theseconditions, one or both of the following two EXAMPLES may provide aneffective treatment.

EXAMPLE 12: Immunodialysis Treatment Primarily for Drug Intoxication

This system is particularly well adapted for the removal of haptens fromthe circulatory system. All of the apparatus, compartments therein andthe materials used with it are of course sterile. In brief, as shown inFIG. 6, a column 25 is divided into a first compartment 29 and a secondcompartment 32 by a semipermeable membrane 33. Such membranes, havingvarious pore sizes, and thus which are permeable to molecules of varioussizes are commercially available. The first compartment 29, an inlet 30for a catheter 30a which is to be connected to an artery 30b of thepatient 30c to be detoxified or otherwise treated, and an outlet 31connected to a catheter 31a which is to be connected to a vein 31b ofthe patient 30c, together comprise a blood flow passage through thecolumn 25. The second compartment 32 includes the heterologous antibodydirected against the hapten which is to be removed. The antibody iscontained in an isotonic solution or other solution such as those usedin conventional dialysis treatment ("artificial kidney"). If the haptenis not known, (as in a patient suffering from an overdose of an unknownpsychoactive drug), the solution in the second compartment 32 mayinclude a mixture of antibodies to drugs which may be implicated. Thesemipermeable membrane is folded or pleated so that the surface area ofinteraction between the patient's blood and the antibody to the drug inthe compartment 32 will be as great as possible. The semipermeablemembrane is chosen to be of such a porosity and permeability as to bepermeable to small molecules like the intoxicating hapten of interest,but is not permeable to large molecules present in the blood of thepatient such as serum proteins.

Because the antibodies specifically bind drug molecules that diffusefrom the blood of the patient in compartment 29 to compartment 32through the semipermeable membrane 33, a continuous gradient is presentfor this intoxicating drug in the patient's blood that thereforecontinues to diffuse from compartment 29 to compartment 32 as long ascompartment 32 includes antibodies directed against the intoxicatingdrug which are free to bind molecules of the intoxicating drug,

The dialysis system, thus far described, has great advantages oversimple blood dialysis, in that it is more specific, and, because of thehigh speed of binding of free hapten by the antibody to it, the systemwill generally require a smaller apparatus, and the time fordetoxification will be much shorter than required by the use of simpledialysis ("artificial kidney"). The chance of saving the patient's lifewill therefore be increased. It may also be possible to use vein to veincatheterization of the patient rather than arterial catheterizationbecause detoxification will be accomplished faster.

When the dialysis system thus far described has been used until theantibody is exhausted for practical use, the solution of theantibody-bound hapten may be removed through a drain 38, and freshantibody solution is added immediately through filler opening 39. Theused antibody-bound hapten may be regenerated for use by the followingsteps: addition of glycine phosphate buffer, pH 2.5 to separate thehapten from the antibody; repeated washings with glycine buffer, eachtime filtering the solution by a positive pressure system through asemipermeable membrane which is permeable to all of the solution exceptthe antibody; addition of PBS buffer, pH 7.4 to the free antibody; andsterilization of the antibody solution by passing it through a bacterialfilter, i.e. one which is permeable to the antibody but not to bacteria.The solution is then ready to be reintroduced to the chamber 32. Thisregeneration of the system makes it far more economical than itotherwise would be.

As shown in FIG. 6, the system may also be automatically regenerated,thereby maintaining its efficiency continuously if desired, and in anycase simplifying the maintaining of the sterility of the system. In thissystem, the hapten-antibody is withdrawn from the second chamber 32 anddelivered to a first chamber 34 of a cleaning column. Glycine phosphatebuffer pH 2.5 is added from a container 40 and differential pressure isapplied to accelerate migration of the free hapten across asemipermeable membrane 35 into a second chamber 24 of the cleaningcolumn. The hapten is drained in solution into a reservoir 36, fordisposal or subsequent recovery and testing. The free antibody solutionis then returned to a pH of 7.4 by the addition of phosphate buffersaline from container 41. The free antibody is then ready to be returnedto the second chamber 32 of the column 25. The entire process ofregenerating the antibody may be controlled automatically by knowndifferential pressure, valving and control equipment.

EXAMPLE 13: Immunoadsorption Treatment

This treatment system is adaptable to the removal of virtually anyreactive species in the blood, but is particularly well adapted to, anddescribed herein with reference to, the removal of haptens, completeantigens, and antibodies by immunological processes. As shown in FIG. 7,the apparatus for this method consists of a column 26 which includes amatrix 37 to which a binding species is linked. The binding species is ahapten, antigen (including a hapten conjugated to a carrier) or antibodywhich reacts specifically with the species which is to be removed fromthe blood. The linkage of the binding species to the matrix 37 maypreferably be directly to the matrix, as when the matrix is made of asynthetic polymer such as polystyrene-latex. The linkage may also bethrough a suitable solid phase coating on the matrix. The antibody isthen linked to the coating by one of the known methods for thepreparation of immunoadsorbents, for example by a modification of one ofthe methods of Campbell (Campbell et al, Proc. Nat. Acad. Sci., U.S.A.,37, p. 575 (1951); Malley and Campbell, J. Am. Chem. Soc. 85, p. 487(1963)). If any chance exists that the solid phase adsorbent may breakloose from the matrix 37, suitable filters are necessary in the system.The matrix may simply be the wall of the (plastic) column if the lengthof the column is sufficient to provide the required surface area forinteraction at the blood-binding species interface. Preferably, thematrix 37 provides a very large surface area. Such matrices include aspiral structure, as shown in FIG. 7, which requires the blood to travelin a thin layer over a large surface. Other matrices include a honeycomb(open porosity) matrix and a fill of plastic beads.

The upper part 27 of the column 26 is connected with tubing to acatheter 27a that is connected to an artery 27b of a patient 27c beingtreated and the lower end 28 of the column is connected to a vein 28a ofthe patient 27c being treated. The blood of the patient flows throughthe column and the species in the blood to which the binding species isspecific becomes bound to the binding species, hence to the matrix.Therefore, the blood which flows from the column 26 is relatively freeof the species sought to be removed.

When the column 26 has been removed from the patient, it may be renewedby elution of the species which is bound to the binding species, byknown techniques, such as washing with glycine phosphate buffer, pH 2.5.

Numerous variations in the materials, devices and methods of thisinvention, within the scope of the appended claims, will occur to thoseskilled in the art in light of the foregoing disclosure.

Having thus described the invention, what is claimed and desired to besecured by Letters Patent is:
 1. An immunoassay method of determining ahapten in a biological sample comprising mixing with said sample a firstantibody to a first site on said hapten and a second antibody to asecond site on said hapten, the quantities of said first and secondantibodies being chosen to produce a precipitate in the presence of atleast a predetermined amount of said hapten.
 2. An immunoassay method ofdetermining a hapten in a sample, the hapten having more than oneantigenic site, the method comprising mixing with said sample a firstantibody to a first site on said hapten and a second antibody to asecond site on said hapten, at least one of said first and secondantibodies being bound to an agglutinable particulate carrier, thepresence of at least a predetermined amount of said hapten in themixture causing agglutination of said particulate carrier.
 3. The methodof claim 2 wherein both said first and second antibodies are bound to anagglutinable particulate carrier.
 4. The method of claim 3 wherein bothsaid first and second antibodies are bound to at least some of the sameindividual particles of said carrier.
 5. The method of claim 2 whereinonly one of the first and second antibodies is bound to an agglutinableparticulate carrier.
 6. A method of determining a hapten comprising astep for mixing a sample with a predetermined quantity of a speciestaken from the group consisting of an antibody to said hapten and anantibody to said hapten bound to an agglutinable particulate carrier,and thereafter combining with said mixture the other of said antibodyand said antibody bound to an agglutinable particulate carrier, saidantibody and said antibody bound to an agglutinable particulate carrierbeing present in quantities to produce agglutination when and only whenthe quantity of hapten in said sample is above a predetermined amount.7. A method of determining a hapten in a biological sample, the haptenhaving more than one antigenic site, the method comprising a first stepof preparing a first antibody to a first antigenic site on the hapten, aseparate second step of preparing a second antibody to a secondantigenic site on the hapten, and thereafter a step of utilizing thefirst antibody and the second antibody to determine the hapten in thesample.
 8. The method of claim 7 wherein the step of utilizing the firstand second antibodies comprises binding said first antibody to anagglutinable particulate carrier.
 9. The method of claim 8 wherein theagglutinable particulate carrier is selected from the group consistingof erythrocytes and latex particles.
 10. The method of claim 8 whereinthe step of utilizing the first and second antibodies comprisesagglutination of a plurality of the particulate carriers when thechemical species is present in a predetermined quantity.
 11. The methodof claim 8 wherein the sample is first exposed to the carrier-boundfirst antibody, and the carrier-bound first antibody is thereafterexposed to the second antibody.
 12. The method of claim 9 wherein thestep of utilizing the first and second antibodies comprises a step ofexposing the sample to the first antibody, and thereafter a step ofexposing the first antibody to the second antibody to determine thepresence of the hapten bound to the first antibody by binding of thesecond antibody to the hapten.
 13. The method of claim 7 wherein thefirst step comprises immunization with the hapten bound to a carrier toprovide steric guidance of the first antigenic site.
 14. The method ofclaim 13 wherein the second step comprises immunization with the haptendifferently bound to the same carrier or bound to another carrier toprovide steric guidance to the second site.
 15. The method of claim 7wherein the first and second steps comprise immunization of differentspecies of animals.
 16. A method of determining a hapten in a biologicalsample, said hapten containing a first antigenic site and a secondimmunogenically different antigenic site, said method comprising a firststep of forming a first antibody to said first site on the hapten, aseparate second step of forming a second antibody to said secondimmunologically different site on the hapten, and a step of utilizingthe first antibody and the second antibody to produce an indication inthe presence of the hapten in the sample.